Cotton is the most extensively used natural fiber in the textile industry. Its annual production worldwide is over 100 million bales, valued at US$45 billion. Cotton lint or seed hair is a terminally differentiated single epidermal cell from 50 species of the genus Gossypium of the family Malvaceae. It is classified as a natural, cellulosic, monocellular and staple fiber. The cultivated cotton varieties, which have been cultivated for more than 5000 years, all come from two diploids (2n=2x=26) (G. herbaceum and G. arboreum) and two allotetraploids (2n=4x=52) (G. hirsutum L., Upland; and G. barbadense L., Sea Island). In 1997 the top five world cotton producers were the United States, China, India, Pakistan and Uzbekstan, producing about 63 million bales.
In the next century, most crops, including cereals, oil crops, fruits, vegetables and other economically important crops, will be genetically engineered with added or modified traits ranging from improvement of yield and quality, to herbicide resistance and pest resistance (Chappell, 1996; Fraley et al., 1986; Herrera-Estrella et al., 1983; Hoekema et al., 1983; Horsch et al., 1985; Jefferson, 1987; Ryals, 1996). In cotton, the new technology will be used to increase yield, improve fiber quality and create new varieties which are resistant to herbicides, pest insects, nematodes and diseases (John, 1996; John & Keller, 1996; John & Stewart, 1992; Murray et al., 1993; Rajasekaran et al., 1996; Schell, 1997; Stewart, 1992).
1. Tissue Culture of Cotton: In 1935, Skovsted reported the first embryo culture of cotton. Beasley (1971) reported callus formation in cotton as an outgrowth from the micropylar end of fertilized ovules on MS medium. Somatic embryogenesis was achieved from a suspension culture of G. klotzschianum (Prive & Smith, 1979). In 1983, Davidonis & Hamilton first succeeded in efficient and repeatable regeneration of cotton (G. hirsutum L.) plants from callus after two-year cultivation. Cotton plants were since regenerated through somatic embryogenesis from different explants (Zhang & Feng, 1992; Zhang, 1994) including cotyledon (Davinonis et al., 1987; Davidonis & Hamilton, 1983; Finer, 1988; Firoozabady et al., 1987), hypocotyl (Cousins et al., 1991; Rangan & Zavala, 1984; Rangan & Rajasekaran, 1996; Trolinder & Goodin, 1988; Umbeck et al., 1987, 1989), stem (Altman et al., 1990; Finer & Smith, 1984), shoot apex (Bajaj et al., 1985; Gould et al., 1991; Turaev & Shamina, 1986), immature embryo (Beasley, 1971; Eid et al., 1973; Stewart & Hsu, 1977, 1978), petiole (Finer & Smith, 1984; Gawel et al., 1986; Gawel & Robacker, 1990), leaf (Finer & Smith, 1984; Gawel & Robacker, 1986), root (Chen & Xia, 1991; Kuo et al., 1989), callus (Finer & McMullen, 1986; Trolinder et al., 1991) and protoplast (Chen et al., 1989).
2. Cotton Transformation: Explants (such as hypocotyl, cotyledon, callus generated from hypocotyl and cotyledon, as well as immature embryos) have been used for Agrobacterium-mediated transformation and particle bombardment (de Framond et al., 1983; Finer & McMullen, 1990; Firoozabady et al., 1987; Perlak et al., 1990; Rangan & Rajasekaran, 1996; Rajasekaran et al., 1996; Trolinder et al., 1991; Umbeck et al., 1987, 1989, 1992). In addition, meristematic tissue of excised embryonic axes has also been used for cotton transformation by particle bombardment (Chlan et al., 1995; John, 1996; John & Keller, 1996; McCabe & Martinell, 1993). Zhou et al. (1983) transformed cotton by injecting DNA into the axile placenta one day after self-pollination. However, cotton transformation is highly dependent on genotype (Trolinder, 1985a, 1985b, 1986; Trolinder & Goodin, 1987, 1988a, 1988b). Apart from a few cultivars which are regeneratable and transformable, such as Gossypium hirsutum cv. Coker 312 and G. hirsutum Jin 7, most other important elite commercial cultivars, such as G. hirsutum cv. D&P 5415 and G. hirsutum cv. Zhongmian 12, are not regeneratable and transformable by these methods.
Based on previous reports and the inventor's own experimental data, high efficiency of callus induction (60%) can be achieved using the hypocotyl as an explant. However, the transformation rate was only 20% (Firoozabady et al., 1987; Umbeck et al., 1987). Several factors can lead to breakthrough of nontransformed calli, or to chimeric calli consisting of predominantly nontransformed cells: (1) low kanamycin levels (a high level of kanamycin is toxic to cotton explants and calli); (2) experience-dependent selection in later stages of callus proliferation; and (3) use of explants such as the hypocotyl which has only partial contact with the selective media (Firoozabady et al., 1987). When the cotyledon was used as an explant, although the transformation rate was higher than that with the hypocotyl, it was often difficult to eliminate Agrobacterium during subsequent culture (Jiao G.-L and Chen, Z.-X., personal communication; Umbeck et al., 1987, 1989). The transformation rate of meristemic tissue through particle bombardment was simply too low (0.02%-0.22%) compared to that of Agrobacterium mediated transformation.
There thus remains a need for methods of producing transgenic cotton plants that provide high rates of transformation along with high rates of transformants among regenerated somatic embryos.